The most abundant organisms in the world's oceans are photosynthetic microbes that are less than ˜20 μm in size. The photosynthetic microbes—the phytoplankton—are believed to generate about 50% of the oxygen and organic matter produced on Earth each year and serve as the base of marine food webs.
Flow cytometry is a technology for counting and/or otherwise examining small particles, for example cells and the like, by passing a stream of fluid in which the particle are suspended through a detection apparatus. In modern flow cytometry systems the detection apparatus typically relies on detecting the optical response produced as the particles pass through an illumination region of the device. In some microbial flow cytometers, for example, individual particles pass through an illumination zone, or region of interest, at a rate on the order of one thousand cells per second. Detectors, gated electronically, measure the magnitude of a pulse representing the light scattered (and/or fluoresced) by the cells. The pulse magnitudes, or other properties, can then be processed to characterize the cells according to a particular parameter of interest. For example, the angular dependence of scattered light may provide information on the nature of the scattering particles. More importantly, in some applications the fluorescent properties of the detected particles, which may be caused by appropriate fluorophores added to the suspension, provide desired parametric information. Modern flow cytometers use coherent light to illuminate a region of interest in a carefully prepared fluid stream, and collect scattered and/or fluoresced light from particles in the stream to count and characterize the particles.
In recent years, flow cytometers have become an essential tool for oceanographic research. Flow cytometry led to the discovery of Prochlorococcus, believed to be the most abundant photosynthetic organism on Earth. Modern cytometry systems are able to rapidly and accurately characterize the abundance, size, and fluorescent characteristics of individual phytoplankton cells in a fluid stream.
The complexity of flow cytometry and the multi-parametric data it produces has hampered the application of flow cytometry to autonomous platforms. It would be advantageous to expand autonomous measurements of phytoplankton communities by developing a miniaturized low-power flow cytometer. Current underwater flow cytometer systems depend on a supply of clean water for instrument operation. In U.S. Pat. No. 8,773,661, to Swalwell, which is hereby incorporated by reference in its entirety, a virtual core technology is disclosed that enables direct cytometric measurements on a flow of raw seawater. This technology has been successfully implemented in a shipboard-based continuous flow cytometer.
The present inventor has developed a novel cytometer configured to autonomously and continuously measure the abundance and composition of microbial populations, for example phytoplankton cells (0.5-20 μm in size), in surface waters while underway aboard a ship. The cytometer is referred to as the SeaFlow cytometer. The system was designed for phytoplankton because these small cells dominate open ocean environments where the most abundant phytoplankton are Prochlorococcus (0.5 diameter), Synechococcus (2 μm diameter), other picoplankton (<2 μm in diameter), and nanoplankton (2-20 μm diameter). Shipboard, underway measurements were emphasized because it has been shown repeatedly that accurate descriptions of microbial community distribution and abundance require observations that occur at a higher frequency than the rate of cell division or mortality, or the rate of environmental changes. A defining feature of SeaFlow is an optical system development known as virtual core flow cytometry. Virtual core flow cytometry detects particles suspended throughout a relatively larger stream, and discriminates between signals obtained from particles that are in a relatively narrow region of interest, i.e., in a focus region for the optical system, and particles that are not in focus. Virtual core flow cytometry eliminates the need to produce a very small diameter stream with suspended particles that is surrounded by a clear sheath fluid. In the SeaFlow cytometer, measurements are performed within a 200 μm diameter stream of 100 μm filtered seawater, which enables the taking of continuous cytometric measurements.
For more widespread use of virtual core cytometry systems, and in particular to the integration of such systems in autonomous platforms, improvements in reliability, lower costs, smaller size, and reduced power consumption are desirable.